Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies. 

Background: Transcriptome assembly from RNA-sequencing data in species without a reliable referencegenome has to be performed de novo, but studies have shown that de novo methods often have inadequateability to reconstruct transcript isoforms. We address this issue by constructing an assembly pipeline whosemain purpose is to produce a comprehensive set of transcript isoforms.

Results: We present the de novo transcript isoform assembler ClusTrast, which takes short read RNA-seq dataas input, assembles a primary assembly, clusters a set of guiding contigs, aligns the short reads to the guidingcontigs, assembles each clustered set of short reads individually, and merges the primary and clusterwiseassemblies into the final assembly. We tested ClusTrast on real datasets from six eukaryotic species, andshowed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novoassemblers, at a moderate reduction in precision. For recall, ClusTrast was on top in the lower end ofexpression levels (<15% percentile) for all tested datasets, and over the entire range for almost all datasets.Reference transcripts were often (35–69% for the six datasets) reconstructed to at least 95% of their length byClusTrast, and more than half of reference transcripts (58–81%) were reconstructed with contigs that exhibitedpolymorphism, measuring on a subset of reliably predicted contigs. ClusTrast recall increased when using aunion of assembled transcripts from more than one assembly tool as primary assembly.

Conclusion: We suggest that ClusTrast can be a useful tool for studying isoforms in species without a reliablereference genome, in particular when the goal is to produce a comprehensive transcriptome set withpolymorphic variants.

The sequence assembly problem, known to be NP-hard, is one of the fundamental problems in bioinformatics, not the least when studying genomes or transcriptomes. Several different methods have been proposed, both with and without reference guidance, but most of these methods are memory intense.

Here, we attempt to prove the concept of applying Ant Colony System (ACS), a swarm intelligence meta-heuristic, to the problem of de novo transcriptome assembly. First, we show how the assembly problem can be reduced to a set of established NP-hard problems, where ACS has previously been applied. Next, we develop an ACS-based de novo transcriptome assembler. Finally, we benchmark the novel assembler with a set of newly developed and/or widely used transcriptome assemblers and find that the ACS approach provides the most memory efficient assembly of the tested methods, using at most 49\% of the memory required by any other method, with a moderate reduction in precision and an appreciable reduction in recall.  This suggests that memory efficient de novo transcriptome assembly using ACS is a working concept,, where further improvements are desired. The ACS approach may potentially also be useful in other assembly areas.

The gene families MADS-box, FT and SPL are all known to be related to cone setting inconifers. Since the genome of most conifers are large and repetitive, no full in-depth studyof these gene families (or any other gene family in conifers) has been possible.

With the advent of a new, near-complete, reference genome assembly of Picea abies, weperformed an in-depth analysis of these gene families, in order to find novel genes withinthese families and simultaneously verify the quality of the newly assembled reference genomeand of its corresponding annotation. We used 30 Illumina and two PacBio RNA-seq datasetsfrom cone setting studies and assembled the corresponding transcriptomes, from which weidentified 35 novel MADS-box genes, 5 novel SPL-genes and 5 novel FT-genes among theannotated genes. We also verified the existence of an exon in the MADS-box gene DAL19,previously only detected by assembly, and we pinpointed the genomic location of five previ-ously identified MADS-box genes missing in the new annotation. This study leaves us withthe same number of MADS-box and SPL-genes as was annotated in a recently publishedreference genome in another conifer, Pinus tabuliformis.

The conifer Norway spruce (Picea abies) has an uneven cone-setting and initiates cones onlyevery 3rd to 5th year. The cone-setting is in large synchronised on the population level,resulting in good or bad cone-years. However, variation in cone-setting frequencies andnumber of cones produced per tree can be observed among different genotypes. Here, we aimto study the genetic basis for the natural variation in cone-setting ability. To this end, we haveset up a field trial consisting of 560 ramets representing eighteen P. abies genotypes and havemonitored the cone-setting in these trees since 2008. Using this material, we study the naturalvariation in regulatory elements of a key cone-setting regulator, Picea abies SQUAMOSABINDING PROTEIN LIKE1 (PaSPL1). We have previously established that the combinedaction of miRNA156 and miRNA529 negatively regulates PaSPL1. Here, we use massivelyparallel sequencing to study the expression of these and other microRNAs in the P. abiesgenotypes. While no direct link can be established between miRNA expression levels and cone-setting frequencies, we report on individual genotype differences that grade the relativeimportance of miR156 and miR529 in regulating PaSPL1 transcript levels. We also clone, andSanger sequence the putative promoter of PaSPL1 from the genotypes in the field trial. Byanalysing variation in SNP data, we identify putative Gibberellic Acid (GA) responsiveelements enriched in the genotype with the highest cone-setting frequency. As a complement,we also sequence the transcriptomes of three genotypes before and after GA-inductions. Thisanalysis shows that relatively more genes are activated in a low cone-setting genotype than ina high cone-setting genotype. We also demonstrate that GA-regulated MYB transcriptionfactors are upregulated upon GA induction, providing a possible link between GA-inducedcone-setting and the regulation of PaSPL1 that merits further studies.